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Sur Lie Science – Wine Character Revealed

Peter Salamone, PhD

Technical Manager, North America Laffort USA

Presented at Brock University – CCOVI February 6 2013

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Sur Lie Ageing - Batonnage

Sur Lie is the French term for leaving the wine in contact with its lees

Batonnage is the term for stirring the lees back up into the wine

Classical French Burgundian schedule for sur lie cellar ageing

Rack off gross lees – “debourbage” – Nov/Dec Rack again in March

Rack again in June – SO2 add Rack in Sept followed by cellar ageing/bottling

The Roman historian Cato is credited with observing that wines left on their lees developed different flavors than those racked clean

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Using Lees to Drive Wine Style

Observed Benefits of Sur Lie Ageing enhance structure and mouthfeel extra body, decreased astringency increase aromatic complexity flavor-aroma depth and length increase perception of sweetness

increased color stability increased protein stability increased tartrate stability oxidation protection

improve nutrition for MLF improved fining and clarity

What Risks are Involved? reductive aromas – H2S, mercaptans wine oxidation from frequent stirring

microbial sanitation inhibition of MLF

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Yeast Autolysis PEPTIDES – FATTY ACIDS

NUCLEOTIDES

AMINO ACIDS

POLYSACCHARIDES

Yeast autolysis occurs at the end stage of alcoholic fermentation and beyond when physical pressure, hydrolytic enzymes and oxidative damage degrade

yeast cell integrity releasing cellular components into the wine

• flavor-aroma

• sweetness

• nutrients

• anti-oxidation

• stability

• mouthfeel

• Anti-oxidation

• fining

• flavor-aroma

• nutrients

• flavor-aroma

• nutrients

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Yeast Schematic Diagram

Cell Wall Mannoproteins Cell Membrane Associated Peptides Cytosolic Peptides and S- amino acids Cell Wall-Membrane Fragments

Other molecules will probably be very interesting for winemaking as well…

Yeast Derived Molecules from Sur Lie

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Yeast Cell Wall and Membrane

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Sur Lie Research Initiative

Laffort Pillars for Growth

Research Innovation Quality

Todays Focus

Peptides in Wine Mannoprotein Characteristics Anti-Oxidation and Fining

Denis Dubourdieu Philippe Marullo Marie-Laure Murat T. Van der Westhuizen Maryam Ehsani

Virginie Moine Alex Marchal Ann Hebert Paul Boyer Charlotte Gaurroud

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Peptides in Wine

The aims of the present investigation were first to validate the role of yeast lees on the increase of sweetness empirically observed during the autolysis process

and then to identify the chemical or biochemical origin of this phenomenon

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Validation of the observation of sweetness in lees

Wine base was red wine 12.2% alc, 6.9 g/l glycerol, 0.37 g/l g+f Lees generated by yeast harvest and placement in red wine base

Forced Ranking Sensory Test

Comparison of ethanol concentrations

Comparison of glycerol concentrations

Comparison of increasing amounts of lees

Perception of Sweetness in Lees

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Validation of Sweetness in Lees

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Yeast Lees Autolysis Medium

YLAM prepared to simplify purification 1) Saccharomyces grown in defined medium

2) Cells harvested, washed and resuspended

3) Model Solution autolysis for 10 days at 32°C in dark

4) Autolysate subjected to ultrafiltration

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Fermented model medium

Retentate >10 KDa

Filtrate

Ultrafiltration vs 10 Kda filter

Ultrafiltration vs 3 Kda filter

Nanofiltration vs 0.5 Kda filter

Filtrate Retentate 10-3 KDa

Retentate 3-0.5 KDa

Filtrate < 0.5 KDa

Fractionation protocol

Membrane Filtration of YLAM

5 Fractions for testing 5

1

2

3

4

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Sensory Analysis of UF Fractions

In triangle testing only YLAM preparation and 0.5-3.0 kDa retentate showed significant differences in sweetness perception

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Proteinase K Digestion

Enzymatic treatment investigating the peptide nature of the sapid effect 1) Concentrated solution of sapid fraction

2) Treatment with Proteinase K

3) Sensory evaluation

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Proteinase K Digest Evaluation

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HPLC Peptide Purification

A Peptide HR column B RP-18 column

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sp|P22943|HSP12_YEAST 12 kDa heat shock protein (Glucose and lipid-regulated protein) – Saccharomyces K.ADKVAGKVQPEDNK.G 1498.78600 K.EYITDKADKVAGKVQPEDNK.G 2248.14557 K.ASEALKPDSQK.S 1173.61099 D.AVEYVSGRVHGEED.P 1546.71323 K.ASEALKPDSQKSYAEQGKEYITDK.A 2686.32063 Y.VSGRVHGEEDPTKK. 1538.79215 K.ADKVAGKVQPED.N 1256.64811 K.ASEALKPDSQKSYAEQGK.E 1936.96106 D.AVEYVSGRVHGEEDPTKK. 2001.00359 K.ADKVAGKVQPEDNKGVFQGVHD. S2338.17860 K.GVFQGVHDSAEKGKDNAEGQGESLADQAR.D 3000.40419 sp|P00560|PGK_YEAST Phosphoglycerate kinase (EC 2.7.2.3) - Saccharomyces cerevisiae (Baker's yeast) K.RVFIR.V 690.44095 D.KISHVSTGGGASLE.L 1342.69612 E.VVKSSAAGNTVIIGGGDTATVAKK.Y 2244.25579 K.SSAAGNTVIIGGGDTATVAKK.Y 1918.02400 R.IVAALPTIK.Y 925.60808 sp|P00924|EAsnO1_YEAST Enolase 1 (EC 4.2.1.11) (2-phosphoglycerate dehydratase) (2-phospho-D- glycera A.GENFHHGDKL.- 1153.53850 F.AGENFHHGDKL.- 1224.57561 Y.ARSVYDSRGNPTVE.V 1550.75576 V.SLAASRAAAAEKNVP.L 1455.79142 sp|P00950|PMG1_YEAST Phosphoglycerate mutase 1 (EC 5.4.2.1) (Phosphoglyceromutase 1) (PGAM 1) (MPGM D.PEAAAAGAAAVANQGKK.- 1524.81288 R.AIQTANIALEK.A 1171.66811 Y.YLDPEAAAAGAAAVANQGKK.- 1915.98722 sp|P02994|EF1A_YEAST Elongation factor 1-alpha (EF-1-alpha) (Translation elongation factor 1A) (Euk K.AGVVKGKTLLEA.I 1185.72015 Y.KIGGIGTVPVGR.V 1153.70517 sp|P00445|SODC_YEAST Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) - Saccharomyces cerevisiae (Baker's-. VQAVAVLKGDAGVSGVVK.F 1696.99560 sp|P32340|NDI1_YEAST Rotenone-insensitive NADH-ubiquinone oxidoreductase, mitochondrial precursor S.KNLYSNKRLLTSTN.T 1651.91259 sp|P05743|RL26A_YEAST 60S ribosomal protein L26-A (YL33) - Saccharomyces cerevisiae (Baker's yeast) R.RVLLSAPLSK.E 1083.68846 tr|Q07653|Q07653_YEAST S.cerevisiae chromosome IV reading frame ORF YDL223c - Saccharomyces cerevis K.ANAKVLEEDAPGYKR.E 1589.82820

Peptide Sequencing Results

Online Capillary HPLC Nanospray Ion Trap

MS/MS Analysis

BLAST Search for ID of Peptides

Majority of isolated and

identified peptides were from Hsp12

HYPOTHESIS Hsp12 peptide source

of sweetness

TEST: Genetic Knockout

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Yeast Strains and Genetics

1) Saccharomyces strain FX-10 is a homothallic, fully homozygous diploid strain

2) Create haploid strain

3) Use Cre-Lox recombination to KO Hsp12

4) Cross ∆Hsp12 with FX-10 by spore micromanipulation

5) Segregate and allow self diploid formation (HO endonuclease)

6) Verify homozygous ∆Hsp12 by sporulation on selective media and PCR

Micromanipulator

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Yeast Strains and Genetics

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Evaluation of a ∆Hsp12 Strain

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Sensory Validation of Sapid Effect of Lees not ethanol or glycerol

Biochemical Determination of Sapid Molecule protein nature shown by digestion

Purification and Identification of Sapid Peptide 2 HPLC separations, LC-MS ID, BLAST

Genetic Validation of Sapid Peptide Source ∆Hsp12 Saccharomyces constructed

Summary of Investigation

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Mannoproteins in Wine

Mannostab: The Award Winning New Potassium Bitartrate Stabilisation Product Boyer, P.K., Moine-Ledoux, V.

Australia & New Zealand Grapegrower & Winemaker June 2007; 57-62

Role of Yeast Mannoproteins in Tartrate Stability of Wines Dubourdieu, D., Moine-Ledoux, V.

1997 Rev. Oenol., 85:17

• Gold Innovation Trophy Vinitech 2006 Bordeaux - France

December 2005 OIV Regulatory Approval of Mannoproteins

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Mannoproteins in Wine

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HPLC Analysis of MP Extracts

Heat extraction profile - MEC Enzyme digestion profile - MEE

void void eluent eluent

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Capillary Electrophoresis Separation

MEC

MEE

minutes

Peak W is clearly a point of differentiation

between the heat treated sample and the enzyme treated sample

Peak W was shown to exhibit the protein and

tartrate stabilization properties

W

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Protein Stability in Wines

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Analysis of MP32

MEC MEE

Only MP32 increased in concentration

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Specific Mannoprotein Effects

Wine

Control

MEC 25 g/hl

MEE 25 g/hl

Rosé 1 White 1 Red 1

*** **** ***

** **** **

0 0 0

Comparison of the tartrate stabilization effect between heat

extracted (MEC) and enzyme extracted mannoproteins (MEE)

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Differential Specificity of MP

Tartrate Stability tested at low temperature

(-4°C for 6 days)

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Analysis of MP40

P1 P2

HPLC separation of MEE

0

0,05

0,1

0,15

0,2

0,25

0,3

0 5 10 15 20 25 30

Purified P1 and P2 fractions by HPLC g/hl

P1 P2 MP40

variation of potassium (g/l)

after cold treatment

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Analysis of MP40

conc.

Only fraction P2 including MP40 allows a stabilization.

Through HPLC and Concanavalin A Affinity

Chromatography Purification the ~40kDa mannoprotein increased

in concentration and effectiveness

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Colloidal Behavior of Mannoproteins

0,05

0,15

0,25

Mannostab® g/hl

0 15 30 100 0

0,1

0,2

0,3

Potassium variation (g/l)

after cooling wine

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Crystalization of Potassium Bitartrate

Structure of the crystal: orthorhombic geometry

Mechanism of Crystallization:

1. Nucleation : formation crystal germ 2. feeding : growing of the crystal

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Microscopic Observation of the Crystallization of Potassium Bitartrate

Control

MP40

27/06 30/06 2/07 4/07 7/07 Date of obs.

With MP40 crystals are flat - undeveloped

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MP40 Mannoprotein Summary

• Naturally present in wine, MP40 is the only mannoprotein having a stabilizing effect regarding tartrate precipitation in wine

• Effective action based on the inhibition of the

crystallization of potassium bitartrate

MP 40 the first natural treatment to stabilize tartrate in wines

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MP40 Winemaking Impact

Quality Improvements Natural Wine Ingredient Preserves Wine Balance

Maintains Color Long Term KHT Stability

Ease of Use

Direct Addition to Wine Rapid Dissolution

Addition can be Automated Rapid Stabilization

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MP40 Winery Impact

Environmental Benefits Reduced Water Use

Reduced Processing Waste Reduced Carbon Footprint

Economic Benefits

Increased Wine Yield Reduced Labor - Time No Capital Investment

Energy Savings Reduced Maintenance Costs

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Tartrate Stabilization by Inhibitors

TARTRATE STABILIZATION

SUSTRACTIVE TECHNIQUES

- Traditional Cold Stabilization -Refrigeration

- Membrane Based Technique (Electrodialysis)

NON-SUBTRACTIVE INHIBITORS

- Yeast Mannoprotein (Natural Inhibitor– MP40)

-Carboxymethyl Cellulose (CMC – Cellulose Gum)

-Metatartric acid

potassium

bitartrate

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CMC Molecular Structure Characteristics

β 1-4 glycosidic linkage – DP – Degree of Polymerization

pKa = 4

pKa = 4

Carboxymethyl groups – DS – Degree of Substitution

Na+

Polymer generated as a Sodium salt – Refinement/Processing reduces Sodium content

DP – Degree of Polymerization Influences - Viscosity, Fluidity DS – Degree of Substitution Influences - Solubility, Efficiency

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CMC Oenological Properties

CMC Interaction Disrupts Bitartrate Crystal Formation

CMC action results in an inhibition of microcrystal growth by disorganization of the 010 surface of the nucleated bitartrate crystal

The negatively charged ionized form of CMC interacts with the (010) face of a bitartrate crystal, specifically the positively charged layer of K+ on the crystal

face

Inhibition of KHT crystal growth by

CMC THK crystal shape without and with CMC

H+

K+

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Chemical Oxidation

Oxidation of Ethanol to Acetaldehyde and Acetic Acid

HYDROXYL (alcohol)

CARBONYL (aldehyde)

CARBOXYLIC ACID

Hydrogen Peroxide

• Fenton Reaction • Fe dependent

• Peroxide reaction product

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Fenton Reaction, Sulfites, Oxygen, Catechols and Quinones – Oh My!

Daniliwicz, AJEV, 62:3 (2011)

Proposed interaction of a catechol and O2 in the presence of sulfite.

Uptake of O2 by polyphenols in model wine in the absence and presence of sulfite

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Redox Potentials

+920 mV

+480 mV

+282 mV

+170 mV

Glutathione

Tocopherol / Vit E

Ascorbic acid / Vit C

SO2

Tannins +600 - 750 mV

SO2 comes from Yeast as well as Winemaker addition Tannins come from Grapes and Oak as well as Winemaker addition Glutathione comes from Grapes as well as Yeast or by Winemaker addition

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Glutathione as an Antioxidant in Wine

ɣ - Glutamylcysteine (GGC)

Glutathione

Glutathione and its precursors added during late fermentation allows yeast to accumulate and release slowly during lees ageing - autolysis

Glutathione added directly to aqueous solution or finished wine can be rapidly oxidized and with no Glutathione Reductase to recycle it loses antioxidant properties

GRP

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Glutathione in Juice and Wine

Glutathione in the juice

(mg/L or ppm)

Glutathione in the corresponding wine

(mg/L or ppm)

9 5 4 17 2

11 7 6 22 3

Valarie Lavigne, 2000

• Glutathione in juice is proportional to the initial YAN • Grape GSH can be rapidly lost by oxidative juice handling • Good AF Nutrition (N/C balance) allows yeast to release additional GSH • GSH in yeast can be supplemented with a timely nutritional addition • 20 ppm+ GSH is needed in finished wine for optimal protection • Recent evidence of Glutathione preservation effect of SO2 in organic wines

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Yeast hulls generated during autolysis with high adsorbing capacity are rich in proteins

Samples reacted with Bradford protein reagent

Albumin Gelatin 1

Gelatin 2

Yeast Hulls 1

Yeast Hulls 2

Reagent changes to blue with protein interaction

After centrifugation proteins are localized in tube bottoms,

ie in the yeast hulls

Selective Adsorption-Yeast Hulls

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Yeast Hull Preparation

Albumin

Potentiel Zeta (mV)

-18,7 mV

+ 31,1 mV

Despite a negative charge, yeast hulls react with tannins by hydrophobicity This action mechanism is different from traditional fining agents

Particles size repartition

Selective Adsorption-Yeast Hulls

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Comparison between albumin and yeast hull fining

0

2

4

6

8

10

12

0 10 20 30 40 50 60 70 80

Albucoll 5 cL/hL

Biolees 20 g/hL

Lees Height

Time in Hours

Lees 5 times more compact than with albumin

Easier racking, less wine loss

Selective Adsorption-Yeast Hulls

Albumin Yeast Hull Preparation

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Continuing Investigations

Lees and Oak Interactions

Lees and MLF Influences

Specific Yeast Cell Wall-Membrane Components and Detoxification

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Research Quote

“The task is...not so much to see what no one has yet seen; but

to think what nobody has yet

thought, about that which everybody

sees.”

Erwin Schrodinger 1933 Nobel Prize for Physics

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Peter Salamone, Ph.D.

Technical Manager

North America

Sur Lie Science – Wine Character Revealed

? Questions – Discussion !

Laffort in Ontario:

Vines to Vintages Inc.

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KHT Stability in Fined Red Wines